FIV causes an AIDS-like syndrome in the domestic cat and represents the smallest natural animal system for study of lentivirus infections. The protease (PR) shares substantial structural homology with PR of HIV-1 but has unique substrate and inhibitor properties, some of which are shared with PRs of drug- resistant HIVs. Studies to date have identified critical amino acid residues of FIV and HIV-1 PRs that are responsible for the observed substrate/inhibitor specificities and mutant FIVs have been prepared that show HIV-like substrate/inhibitor specificities. Furthermore, consensus sequences have been identified that represent optimal cleavage targets for a broad spectrum of FIV and HIV-1 PRs. This information has been used to develop a broad-based inhibitor (termed TL-3) that is efficacious against both FIV and HIV-1 PRs in vitro and virus infection in tissue culture. In this continuation study, we propose to further define substrate/inhibitor interactions by identifying structural changes responsible for escape by drug-resistant HIV-1 and FIV mutants and establishing the limits of protease mutability. Initial experiments have identified a mutant HIV with a 19-fold increase in resistance to TL-3. The PR of this isolate as well as wild-type and other drug resistant PRs have been sub-cloned, over-expressed in E. coli, and purified for enzymatic analyses. These PRs will be employed in conjunction with phage display libraries to select for substrates preferred by drug resistant variant PRs and optimal cleavage sequences will be identified. The information gained may then be used to prepare inhibitors with greater selectivity for drug-resistant PRs. Tests will be performed in tissue culture against both FIV and HIV-1 to monitor inhibitor efficacy, to test for relative viral fitness and to track the course of drug resistance development. Results of the ex vivo studies will be used to select mutant FIVs for tests in vivo, to assess drug sensitivity, viral fitness and resistance development as a function of both drug and immunological pressure in the natural host. These studies will advance our understanding of the structural basis for drug resistance and lead to development of effective inhibitors less susceptible to escape via mutations within PR.